Up-regulation of S100P, a member of the S100 calcium-binding protein family, is an early molecular event in the development of pancreatic cancer and it is expressed at high levels in both precursor lesions and invasive cancer. To gain more insight into the molecular mechanisms underlying the functional roles of this protein, we stably overexpressed S100P in the Panc1 pancreatic cancer cell line and identified the consequent changes in global protein expression by two-dimensional difference in-gel electrophoresis. The observed changes in target proteins were confirmed by Western blot analysis and immunofluorescence, whereas their functional effect was investigated using motility and invasion assays. In this study, we have shown that overexpression of S100P led to changes in the expression levels of several cytoskeletal proteins, including cytokeratins 8, 18, and 19. We have also shown disorganization of the actin cytoskeleton network and changes in the phosphorylation status of the actin regulatory protein cofilin. Additionally, we have shown that overexpression of S100P leads to increased expression of another early pancreatic cancer marker, S100A6, as well as the aspartic protease cathepsin D, both of which are involved in cellular invasion. Functional studies showed that the increased invasive potential of S100P-overexpressing cells was at least partially due to the increase in cathepsin D expression. In summary, our data suggest that these changes could contribute to the metastatic spread of pancreatic cancer and may explain the devastating prognosis of this disease.